No direct means is available currently for assessing the production of acid by osteoclasts. This proposal is a plan to develop and refine such a method. This method should make it possible to screen potential inhibitors of osteoclastic acid production and should help elucidate the mechanism of acid secretion by these cells. Osteoclasts from the endocranial surfaces of the frontal and parietal bones of mice will be used for measurement in situ in organ culture. Seven-day-old animals will be used. Bones will be cultured for at least 4 days prior to beginning any treatment with test agent to allow endogenous hormones to become inactive. The method is based on the uptake of acridine orange, a fluorescent weak base, into acidic cellular compartments. The more acidic the compartment, the more acridine orange is accumulated in it. In preliminary experiments, the fluorescent intensity of osteoclasts was found to increase with the concentration of parathyroid hormone present in the culture medium. Time course studies (12-84 hours, as well as basal) were performed. Nigericin, a K+ ionophore, completely eliminates all fluorescence in osteoclasts. Monensin, a Na+ ionophore, was partially effective in eliminating fluorescence. Acetazolamide is a dose-dependent inhibitor of fluorescence. This method should provide rapid and reproducible means for assessing the effectiveness of inhibitors and stimulators of acid production by osteoclasts. This in turn, may lead to schemes for treating metabolic bone diseases characterized by increased or decreased osteoclastic activity.